P1005: EFFECT OF TL-895, A NOVEL BRUTON’S TYROSINE KINASE INHIBITOR (BTKI), ON ONCOGENIC JANUS KINASE 2-V617F (JAK2VF) SIGNALING
Authors: Subbaiah Chary Nimmagadda, Paulina Marie Budde, Kaiyan Sun, Todd Covey, Cecile Krejsa, Wayne P. Rothbaum, Nikolas von Bubnoff, Cyrus Khandanpour
Journal: HemaSphere (2023)

Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research Background: Myelofibrosis (MF) is a clonal hematopoietic disorder characterized by aberrant JAK2 signaling, megakaryocyte hyperplasia, abnormal cytokine production, and dysregulated trafficking of MF cells to extramedullary sites. While the JAK1/2 inhibitor ruxolitinib (rux) can control symptoms and improve splenomegaly, treatment is often limited by loss of response, adverse events, and blast phase transformation (Palandri 2019). The lack of disease-modifying effects and high rates of discontinuation from rux treatment underscore a need for novel therapies. Activated BTK mediates cell adhesion and chemotaxis toward stromal-cell derived-factor (SDF1α) in JAK2VF cells (Nimmagadda 2019). TL-895 is a highly potent, selective, orally available, small molecule inhibitor of BTK and bone marrow tyrosine kinase X-linked (BMX). TL-895 is currently being investigated in MF patients for its potential to impair stromal adhesion, disrupt aberrant MF cell trafficking, reduce cytokine-mediated symptoms, and improve dysfunctional megakaryopoiesis. Aims: Evaluate the effects of TL-895 on signaling, cell adhesion and chemotaxis in JAK2VF cells. Methods: Murine 32D or BaF3 cells ectopically expressing human JAK2WT and JAK2VF were treated for 4h or overnight with TL-895, ibrutinib (ibr), or rux. Intracellular signaling was evaluated by Western blot. Static adhesion assays were performed using JAK2VF cells treated with TL-895 or rux at 0.25 μM on intercellular adhesion molecule-1 (ICAM) or vascular cell adhesion molecule-1 (VCAM)-coated plates and assessed by fluorimetry. Trans-well motility assays were performed with pre-treated JAK2VF cells (TL-895 at 0.1μM; rux at 0.25μM; ibr at 0.5μM) using SDF1α as the chemo-attractant. RAC1 activation was measured by G-LISATM assay. Results: 32D JAK2VF cells had significantly higher BTK pathway activation than 32D JAK2WT cells, and treatment with either rux (0.25μM) or TL-895 (0.1μM) effectively inhibited BTK activation in JAK2VF cells. Accordingly, in serum-starved 32D JAK2WT cells, TL-895 (0.1μM) or ibr (2μM) also strongly inhibited phospho-BTK after EPO- and SDF1α-stimulation. Constitutive JAK2 signaling in 32D and BaF3 cells significantly increased adhesion to ICAM and VCAM (p<0.05). In static adhesion assays on VCAM- or ICAM-coated surfaces, TL-895 significantly blocked cell adhesion (40% decrease; p<0.001 and p<0.005, respectively). In chemotaxis assays, TL-895 inhibited the migration of JAK2VF cells toward SDF1α (57% decrease; p<0.05). Furthermore, the GTPase RAC1 was activated in JAK2VF cells and TL-895 treatment decreased RAC1 activation (39% decrease; p=0.08). These findings demonstrate that inhibition of BTK activity has a downstream impact on pathways critical for adhesion and chemotaxis in JAK2VF cells. Summary/Conclusion: Increased adhesion and aberrant chemotaxis contribute to MF pathogenesis. The BTK pathway, which plays a key role promoting cell adhesion and chemotaxis, was activated by JAK2-mediated signals and the chemokine SDF1α. TL-895 potently inhibited BTK signaling to reduce cell adhesion and SDF1α-mediated chemotaxis in JAK2VF cells. These data suggest that BTKi treatment with TL-895 could inhibit the interactions of JAK2VF myeloblasts with their microenvironment and may improve patient outcomes in MF, a hypothesis that is being evaluated clinically (NCT04655118, NCT05280509, NCT04640532). Keywords: Myelofibrosis, Chemotaxis, Adhesion, Bruton’s tyrosine kinase inhibitor (BTKi)